Team:Freiburg Bioware/testpage

• Materials

Results of agarose-gel:

• Expected fragments of 3677 bp and 974 bp can be cerified. The insertion of the RFC25_MCS has been inserted.
  
For further verification the insert has to be sequenced has to be conducted (to do for 04.06.2010).

Picking clones of Thymindinkinase of Amor

• • 5 clones of the XL-1 Blue colonies containing the plasmid pUB6/HV5/His6 with the thymidinkinase have been picked from LBamp-agarplates
  • 1 clone of the XL-1 Blue colonies containing the plasmid pUB6/HV5/His6 without the thymidinkinase (control) has been picked from LBamp-agarplates
  • all clones have been inoculated in 10 mL LB containing 10 µL Amp. Incubation: 37°C over-night.
  • to do: Mini-Prep of pUB6/HV5/His6 with the thymidinkinase and pUB6/HV5/His6 without the thymidinkinase (control)

Idea

• Insertion of Kozak consensus sequence before MCS to enhance gene expression (cloning consideration in Stratagene manual)
  • New RFC standard with Kozak sequence for eucaryotes??

21. Labortag 04.06.2010: DKFZ plasmid Retrafos, TK/GMK Mini-Prep

Investigators: Adrian, Bea, Chris W., Hanna


DKFZ

Comments: Plasmids of PD Kleinschmidt of the DKFZ arrived. The DNA was dried on a whatman paper.
Plasmids received:


• pXX6 alle Adenovirus Helfer Gene ohne AAV Gene; Plasmid von J. Samulski; evtl. Anfragen für Benutzererlaubnis
• pKEX-2XL.Rep40 Expressionsplasmide für das Rep Proteine 40
• pKEX-2XL.Rep 52 Expressionsplasmide für das Rep Proteine 52
• pKEX-2XL.Rep 68 Expressionsplasmide für das Rep Proteine 68
• pKEX-2XL.Rep 78 Expressionsplasmide für das Rep Proteine 78
• pCMV-VP(HS) Expressionsplasmid der drei VP Proteine in Kombination (assemly kompetent
• pKEX-VP1 Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
• pKEX-VP2Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
• pKEX-VP3Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
• pTRUF_CMV_eGFPEinzelstrang Vektor zur Expression von eGFP
• dsAAV_CMV_eGFP"self complementary" Expressionsvektor von X. Xiao; evtl. anfragen wegen Benutzererlaubnis
• pTAV2 (gesamtes AAV Genom im "blue script" Vektor)
• pDG komplettes Helferplasmid zur Vektorherstellung; Grimm et al. 1998




• In order to obtain the DNA following steps have been performed:
  • cut out the spot where the DNA is spotted with a clean scalpel (note: scalpel should not have any contamination).
  • put a 0,5 mL Eppi in a 1,5 mL Eppi. Put little holes in the smaller eppi.
  • transfer Whatman paper into Eppi
  • add 50 µL TE-EF (Redissolving buffer) to whatman paper and wait 15 minutes
  • centrifuge eppis at 2000 rpm, 10 minutes
• Transformation with obtained plasmids was performed.

Fusion Enzyme: Thymidinkinase/Guanylate Kinase (TK/GMK)

Plasmid Mini-Prep

• experiment date: 04.06.2010 ; time: 3,5h
• name of investigator: Adrian, Bea, ChrisW.,Hanna
• new vector name: pUB_V5_His6 + TK/GMK (fusionenzyme)

Glycerol Stocks

Given Plasmid-Number

Nanodrop concentration

• Plasmid
  • Given Plasmid-Number: P15; DNA concentration: 493,7 ng/µL ;
  • Given Plasmid-Number: P16; DNA concentration: 464,4 ng/µL;
  • Given Plasmid-Number: P17; DNA concentration: 445,6 ng/µL;
  • Given Plasmid-Number: P18; DNA concentration: 562,9 ng/µL;
  • Given Plasmid-Number: P19; DNA concentration: 528,1 ng/µL;
  • Given Plasmid-Number: P20; DNA concentration: 499,9 ng/µL;

Comments:A Plasmid-Mini Prep with the received Fusionenzyme Thymidinkinase/Guanylate Kinase (TK/GMK)from Amor has been performed.
The DNA will be sent to GATC for sequencing.

• pUB6_V5_His6 - clone 1 (P15) (3 µL added to 27µL H20) has been sent to GATC.
• Expected results: Saturday

22. Labortag 05.06.2010: Insertion of iGEM expression parts into pAAV_MCS

Investigators: Adrian, Bea, Melanie, Hanna

Hybridization:

• Hybridization of received oligos: iGEM expression parts (RFC25 without EcoRI, NotI)
• Prior to opening the tubes, they were centrifugated at 13.000 rpm for 30 sec.
  • expression part MCS_for (charge-no: ST00114065): 108 µL Millipore H_{2</sup>O (Volume on obtained sheet)were added.}
  • expression part MCS_for (charge-no: ST00114066): 165 µL Millipore H_{2</sup>O (Volume on obtained sheet)were added.}
• Resuspended DNA was vortexted.
• Aliquots of both Oligos (1:10) were prepared: 10 µL Oligo + 90 µL H_{2</sup>O (final volume usually 100 µl).}
• Mix together(into PCR-tube):

• Program: ORIGAMI 1 modified for long oligos:
  • 1 99°C 7’
  • 2 99°C 1’
  • -1°C R=0.3 °/s
  • Goto 2 rep 74
  • Hold 4°C

• While hybridization of oligos was performed, digestion of pAAV_MCS vector was conducted
  

following standard protocol for cloning.

Digestion:

• Title: Ligation iGEM expression parts (="iGEM-MCS") with pAAV_MCS
• Plasmid: pAAV_MCS
• Buffer used: 3
• BSA: Yes
• DNA-Concentration: 260 ng/uL
• Restriction-enzyms used:

Enzyme1 (Nr. Lab: 152): ClaI Enzyme2 (Nr. Lab: 15): BglII

• Digestion components :

• Mixture was incubated for 1,5 h at 37°C.

Agarose-Gel:

• 1% agarose gel was prepared, gel ran for 45 minutes(110 V)

• Amount of loading dye added

• Expected size of fragments

</ul>

Gelextraction:

Gel measurement:

• Gelextraction was performed following standard protocol.
• DNA-concentrations were meassured: pAAV_MCS 18.2 ng/µL, Oligos: 136.7 ng/µL -> 1:10 dilution was prepared: 13.67 ng/µL

Ligation:

Trafo was performed (using XL1B cells) following standard protocol.

23. Labortag 07.06.2010: Mini-Preps (Kleinschmidt-plasmids and pAAV_iGEM-MCS)

investigators: Achim, Kira,Jessy, Chris W., Hanna, Adrian, Bea

pAAV_iGEM-MCS

Plasmid Mini-Prep

• experiment date:07.06.2010; time: whole day
• name of investigator: Achim, Kira,Jessy, Chris W., Hanna, Adrian, Bea

Glycerol Stocks

Given Plasmid-Number

Test digestion

• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:___) AgeI ; Enzyme 2 (no.Lab:___) NdeI
• Plasmid
  • Given Plasmid-Number: P34.2; DNA concentration: 433.78 ng/µL ;
  • Given Plasmid-Number: P34.3; DNA concentration: 408.48 ng/µL ;
  • Given Plasmid-Number: P34.4; DNA concentration: 409.80 ng/µL ;

Comments:Clone no.1 was dismissed...

Test digestion:

• Incubation: 45 min, 37°C

Agarose-Gel:

0.5 g Agarose, 50 ml TAE (1%), 3 µL GELRED (3-6µl), at 110 Volt, running time: 45 minutes

• Marker: GeneRuler ladder mix

Kleinschmidt-plasmids

Plasmid Mini-Prep

• experiment date: 07.06.2010 ; time: 10 – 20 h
• name of investigator: Kira, Achim, Jessy, Bea, Adrian, Hanna
• Kleinschmidt-plasmids

Glycerol Stocks

Given Plasmid-Number

Comments: Many things went wrong today!

• Glycerol stocks must be vortexted!
• Check mini-prep buffers - especially buffer PE (needs to contain ethanol!)
• Always check volumes - try to estimate if volume makes sense (check pipettes!)
• Don't discard bacteria cultures until glycerol stocks and mini-preps are successfully done!!!

Today's conclusion: Better ask 2 times than do something wrong without asking!!!

Site-directed mutagenesis of pAAV_iGEM-MCS (PstI)

Quickchange site directed mutagenesis: PCR reaction:

• 2.5 µL 10x Pfu Ultra II buffer
• 0.5 µL template (therefore the a 1:20 dilution of the pAAV_iGEM-MCS (433 ng/µL) was prepared) = 10.825 ng
• 0.56 µL primer 1 (of 1:10 dilution)
• 0.56 µL primer 2 (of 1:10 dilution)
• 1 µL DMSO (primers form very strong secondary structures)
• 0.5 µL dNTP
• 18.88 µL dH_2</sup>O
• 0.5 µL PfuUltra II fusion (1.25 U)

-> end volume: 25 µL

PCR program:
1 x : 2' 95°C (HotStart polymerase) 20 x : 30 s 95°C -> 1' 55°C -> 5' 68°C 1 x : 4°C (over night)
Experiment will be continued tomorrow.